Phage display and combinatorial chemistry are two high throughput approaches for identification of targeted therapeutics of cancer. However, a phage display library has a limited structural diversity and small molecules generated by combinatorial chemistry generally cannot be pooled in large numbers for efficient screening. Our long term goal is to combine the rapid-screening feature of phage display and the diversity- generating power of synthetic chemistry to assemble approaches for high throughput identification of small- molecule antiangiogenic agents and apply these molecules to cancer diagnosis, profiling, imaging, and therapy. The primary objective of this particular application is to develop methods for constructing phage display libraries with expanded chemical diversities and screening these libraries to identify ligands specific for vascular endothelial growth factor (VEGF), a key target of antiangiogenic drugs. Our central hypothesis is that the chemical diversity of a phage display library can be significantly expanded by genetically incorporating two different noncanonical amino acids (NAAs) into the library, varying the identities of NAAs, and/or chemically modifying the incorporated NAAs. The rationale for the proposed research is that, once these unnatural phage display libraries are constructed, a large variety of unnatural peptides with diversified chemical structures can be rapidly screened against many cancer therapeutic targets, leading to identification of new therapeutics for cancer treatment and prevention. Guided by strong preliminary data, the objective of this application will be attained by pursuing three specific aims: 1) Optimize M13KE phage for the unbiased display of 20 natural amino acids and 2 NAAs; 2) Construct unnatural phage display libraries incorporated with different combinations of 2 NAAs and screen these libraries to identify VEGF-specific ligands; and 3) Expand the chemical diversity of unnatural phage display libraries by expanding the pool of genetically encoded NAAs and selectively modifying the incorporated NAAs. The research proposed in this application is innovative, because it will expand the phage display technique significantly by amending the displayed peptides with diversified structure moieties desirable for drug discovery. The proposed research is significant because it will accelerate the current drug discovery progresses and contribute to fulfilling the NIH mission in promoting health and combating diseases.